Driver mutations in Isocitrate Dehydrogenase 1 (IDH1) are present in 70-80% of grade II and III gliomas, which the majority eventually progress to glioblastoma multiforme (GBM). In this molecularly distinct class of malignant gliomas, mutant IDH1 enzyme produces 2-hydroxyglutarate (2-HG), an oncometabolite that inhibits a-ketoglutarate dependent histone and DNA demethylases resulting in characteristic hypermethylation of genomic DNA and suppression of cellular differentiation. We have demonstrated the preclinical efficacy and mechanism of action of the FDA approved DNA demethylating drug 5-azacytidine. In molecularly accurate models, systemic 5-azacytidine administration reduces tumor burden, extends survival and induce differentiation in vivo. For this work we have created our own patient derived model of IDH1 mutant anaplastic astrocytoma, and have additionally obtained models of IDH1 mutant grade III oligodendroglioma and oligoastrocytoma from our collaborators. The focus of this grant is to optimize and understand the mechanism of DNA demethylation therapy for the treatment of IDH1 mutant glioma. In Aim 1 we propose to demonstrate the mechanism and efficacy of 5-azacytidine induced tumor regression in orthotopic IDH1 mutant glioma systems beyond our preliminary findings. In preclinical modeling, we will optimize the mode of administration, confirm intracranial delivery, and survival benefit. To confirm the in vivo mechanism we will show target inactivation, differentiation, and key alterations in transcription and methylation. In Aim 2 we will confirm the synergistic benefit of combining 5-azacytidine with the current standard of care for high grade gliomas. Additionally, we will assess the mechanism of tumor regression in these tumors including alterations in apoptosis and differentiation. In Aim 3 we will perform a preclinical evaluation of synergistic combination of 5-azacytidine with IDH1 mutant protein inhibitors which are currently in clinical trials. Here we will determine if we can achieve a faster and more favorable therapeutic response by halting pathogenic 2-HG production while reversing pathogenic hypermethylation. Additionally, we will assess in depth the mechanism of successful therapy for inhibition of mutant IDH1 protein plus demethylation by 5- azacitidine. Overall, our goal is to elucidate the mechanism of 5-azacytidine induced tumor regression in IDH1 mutant tumors and to ascertain the most effective therapeutic strategy in vivo. Our preliminary results are promising, but optimization of drug administration, synergistic combinations as well as a more in-depth and mechanistic approach is needed to increase the chances that the work will translate successfully to the clinic. The results from this work will allow us to better design and possibly support a new trial for patients with IDH1 mutant glioma, which account for a substantial fraction of brain cancer mortality.